Human Bladder Cancer Cell Lines Research Articles

Diagnosis of bladder cancer using 18F-labeled α-methyl-phenylalanine tracers in a mouse model.

Although 2-18F-fluoro-2-deoxy-glucose (18F-FDG) has established roles in the diagnosis of a variety of cancers, it has limited value in the detection of primary/recurrent lesions in the bladder, mainly because of interference by the pooled radioactivity in the urine. Our previous study revealed promising properties of L- and D-2-18F-α-methyl-phenylalanine (2-18F-FAMP) as radiotracers; i.e., their rapid blood clearance and low renal accumulation. In the present study we evaluated the utility of L- and D-2-18F-FAMP for imaging bladder cancer in a mouse model. We used the human bladder cancer cell line HT1376 to prepare a bladder cancer xenograft model in mice bearing both orthotopic and subcutaneous tumors. Biodistribution and PET imaging studies were performed at 1 and 3h after injection of L-2-18F-FAMP or D-2-18F-FAMP. 18F-FDG was used as a control. At 1h after injection, greater accumulations of both L-2-18F-FAMP and D-2-18F-FAMP were observed in the orthotopic tumors compared to 18F-FDG. The orthotopic tumor-to-muscle ratio of D-2-18F-FAMP was significantly higher than that of 18F-FDG (p < 0.01), because of the rapid blood clearance of D-2-18F-FAMP. L-2-18F-FAMP showed the highest subcutaneous tumor-to-muscle ratio (p < 0.01) due to its high subcutaneous tumor uptake. Compared to L-2-18F-FAMP, D-2-18F-FAMP exhibited faster clearance and lower kidney accumulation. In the PET imaging studies, L- and D-2-18F-FAMP both clearly visualized the orthotopic bladder tumors at 1h after injection. Our study showed that L-2-18F-FAMP and D-2-18F-FAMP have the potential to detect bladder cancer.

GRHL2 Acts as an Anti-Oncogene in Bladder Cancer by Regulating ZEB1 in Epithelial-Mesenchymal Transition (EMT) Process.

PurposeGRHL2 played important roles in different cancers. In this study, we aimed to investigate the roles of GRHL2 in bladder cancer.MethodsThe immunohistochemistry assay was performed to detect the expression of GRHL2 in bladder cancer tissues and adjacent noncancerous tissues and the expression levels of GRHL2 and zinc finger E-box binding homeobox (ZEB1) mRNA in tissues were determined by qRT-PCR. In addition, qRT-PCR and Western blotting were applied to detect the expression levels of GRHL2 and ZEB1 in bladder cancer cell lines (RT4, BIU-87, 5637, T24) and immortalized human bladder epithelial cell line (SV-HUC-1). The cell models with up-regulated and down-regulated expression of GRHL2 were constructed using bladder cancer cell lines T24 and 5637 to investigate the underlying roles of GRHL2 on the proliferation, migration, invasion and EMT process of bladder cancer cells. After that, cell proliferation was evaluated by CCK8 assay, cell cycle assay and colony formation assay. Transwell assay and wound healing assay were performed to determine the invasion and migration ability of the bladder cancer cells. The expressions of epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, Vimentin, Slug and Snail) were assessed by Western blot analysis. Moreover, ZEB1 and GRHL2 were co-transfected into T24 and 5637 cells and their effects on EMT process and invasive capacity of cells were examined.ResultsThe expression of GRHL2 was down-regulated in bladder cancer tissues and human bladder cancer cell lines compared with the normal bladder tissues and immortalized human bladder epithelial cell line. Besides, down-regulation of GRHL2 improved the proliferation ability of bladder cancer cells and promoted the EMT process through up-regulation of ZEB1. The overexpression of ZEB1 partially reversed the inhibitory effect of GRHL2 on EMT.ConclusionGRHL2 acts as an anti-oncogene to regulate bladder cancer cell proliferation and inhibit EMT by targeting ZEB1. This study may provide a theoretical basis for further research.

Oncogenic role of MIR516A in human bladder cancer was mediated by its attenuating PHLPP2 expression and BECN1-dependent autophagy

ABSTRACT Although MIR516A has been reported to be downregulated and act as a tumor suppressor in multiple cancers, its expression and potential contribution to human bladder cancer (BC) remain unexplored. Unexpectedly, we showed here that MIR516A was markedly upregulated in human BC tissues and cell lines, while inhibition of MIR516A expression attenuated BC cell monolayer growth in vitro and xenograft tumor growth in vivo, accompanied with increased expression of PHLPP2. Further studies showed that MIR516A was able to directly bind to the 3′-untranslated region of PHLPP2 mRNA, which was essential for its attenuating PHLPP2 expression. The knockdown of PHLPP2 expression in MIR516A-inhibited cells could reverse BC cell growth, suggesting that PHLPP2 is a MIR516A downstream mediator responsible for MIR516A oncogenic effect. PHLPP2 was able to mediate BECN1/Beclin1 stabilization indirectly, therefore promoting BECN1-dependent macroautophagy/autophagy, and inhibiting BC tumor cell growth. In addition, our results indicated that the increased autophagy by attenuating MIR516A resulted in a dramatic inhibition of xenograft tumor formation in vivo. Collectively, our results reveal that MIR516A has a novel oncogenic function in BC growth by directing binding to PHLPP2 3′-UTR and inhibiting PHLPP2 expression, in turn at least partly promoting CUL4A-mediated BECN1 protein degradation, thereby attenuating autophagy and promoting BC growth, which is a distinct function of MIR516A identified in other cancers. Abbreviation: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BAF: bafilomycin A1; BC: bladder cancer; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CUL3: cullin 3; CUL4A: cullin 4A; CUL4B: cullin 4B; IF: immunofluorescence: IHC-p: immunohistochemistry-paraffin; MIR516A: microRNA 516a (microRNA 516a1 and microRNA 516a2); MS: mass spectrometry; PHLPP2: PH domain and leucine rich repeat protein phosphatase.

Luteolin suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway.

Luteolin is a natural flavonoid with strong anti–oxidative properties that is reported to have an anti–cancer effect in several malignancies other than bladder cancer. In this study, we describe the effect of luteolin on a human bladder cancer cell line, T24, in the context of the regulation of p21, thioredoxin‐1 (TRX1) and the mechanistic target of rapamycin (mTOR) pathway. Luteolin inhibited cell survival and induced G2/M cell‐cycle arrest, p21 upregulation and downregulation of phospho(p)‐S6, which is downstream of mTOR signaling. Luteolin also upregulated TRX1 and reduced intracellular reactive oxygen species production. In a subcutaneous xenograft mouse model using the rat bladder cancer cell line, BC31, tumor volumes were significantly decreased in mice orally administered luteolin compared to control. Immunohistochemical analysis revealed that increased p21 and decreased p‐S6 expression were induced in the luteolin treatment group. Moreover, in another in vivo N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN)‐induced rat bladder cancer model, the oral administration of luteolin led to a trend of decreased bladder tumor dimension and significantly decreased the Ki67‐labeling index and p‐S6 expression. Furthermore, the major findings on the metabolism of luteolin suggest that both plasma and urine luteolin‐3ʹ‐O‐glucuronide concentrations are strongly associated with the inhibition of cell proliferation and mTOR signaling. Moreover, a significant decrease in the squamous differentiation of bladder cancer is attributed to plasma luteolin‐3ʹ‐glucuronide concentration. In conclusion, luteolin, and in particular its metabolized product, may represent another natural product‐derived therapeutic agent that acts against bladder cancer by upregulating p21 and inhibiting mTOR signaling.

Overexpression of Karyopherin Subunit alpha 2 (KPNA2) Predicts Unfavorable Prognosis and Promotes Bladder Cancer Tumorigenicity via the P53 Pathway.

BackgroundWe sought to investigate the expression of KPNA2 in bladder cancer (BC) and its relationship with prognosis, and to analyze the potential mechanism of KPNA2 in promoting BC progression.Material/MethodsThe RNA-seq data on BC from The Cancer Genome Atlas (TCGA) database were imported into R statistical software for differential analysis. The clinical data for patients with BC were screened and analyzed with R software. The survival curve was drawn with the Kaplan-Meier Plotter. The expression of KPNA2 in 4 human BC cell lines and a human bladder epithelial cell line was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The proliferation of BC cells was detected with Cell Counting Kit-8 (CCK8), detection of apoptosis, and flow cytometry, and the migration and invasion of BC cells were detected through Transwell assays. WB was used to detect proteins involved in the P53 pathway.ResultsThe expression of KPNA2 was higher in BC. The difference in KPNA2 expression was associated with many clinicopathological factors, and high expression of KPNA2 was associated with shorter survival time. After KPNA2 knockout, the proliferation, migration, and invasion ability decreased significantly, the cell cycle was clearly arrested in the G0/G1 phase, and the number of apoptotic cells increased. Moreover, CyclinD1, BCL2, and pro-caspase3 decreased significantly, whereas P53, P21, BAX, and cleaved-caspase3 increased significantly. The results in the overexpression group were the opposite of results in the knockdown group.ConclusionsKPNA2 is an oncogenic factor that facilitates BC tumorigenicity through the P53 pathway.

Linking NRP2 With EMT and Chemoradioresistance in Bladder Cancer.

Neuropilin-2 (NRP2) is a prognostic indicator for reduced survival in bladder cancer (BCa) patients. Together with its major ligand, vascular endothelial growth factor (VEGF)-C, NRP2 expression is a predictive factor for treatment outcome in response to radiochemotherapy in BCa patients who underwent transurethral resection. Therefore, we investigated the benefit of combining cisplatin-based chemotherapy with irradiation treatment in the BCa cell line RT112 exhibiting or lacking endogenous NRP2 expression in order to evaluate NRP2 as potential therapeutic target. We have identified a high correlation of NRP2 and the glioma-associated oncogene family zinc finger 2 (GLI2) transcripts in the cancer genome atlas (TCGA) cohort of BCa patients and a panel of 15 human BCa cell lines. Furthermore, we used in vitro BCa models to show the transforming growth factor-beta 1 (TGFβ1)-dependent regulation of NRP2 and GLI2 expression levels. Since NRP2 was shown to bind TGFβ1, associate with TGFβ receptors, and enhance TGFβ1 signaling, we evaluated downstream signaling pathways using an epithelial-to-mesenchymal transition (EMT)-assay in combination with a PCR profiling array containing 84 genes related to EMT. Subsequent target validation in NRP2 knockout and knockdown models revealed secreted phosphoprotein 1 (SPP1/OPN/Osteopontin) as a downstream target positively regulated by NRP2.

Down-regulation of CIT can inhibit the growth of human bladder cancer cells

ObjectiveOur study is to examine the citron rho-interacting, serine/threonine kinase 21 (CIT) in bladder cancer. MethodsWe examined CIT level in human bladder cancer tissues by immunohistochemical staining. To explore the impact of CIT on cell proliferation and apoptosis, we down-regulated its expression in two human bladder cancer cell lines, 5367 and T24. We examined cell growth in 5367 and T24. We also performed in vivo analysis using T24 cells. We further used microarray expression profiling to investigate genes differentially expressed in T24 cells with CIT down-regulated. ResultsIn 100 human samples, CIT was expressed by only 2 of 30 (6.7 %) controls in bladder tissues, whereas by 64 of 70 (91.4 %) cancer patients in tumor tissues (p < 0.001). in vitro analysis demonstrated that CIT knockdown represses cell proliferation by 50 % in both cells and colony formation (77 ± 5 vs. 13 ± 2, p = 0.001 for T24, 58 ± 3 vs. 1 ± 1, p < 0.001 for 5637). We also found CIT knockdown could induce cell cycle arrest, and promote apoptosis in both cells. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces tumor volume (668.4 ± 333.0 vs. 305.7 ± 170.4 mm3, p = 0.02) and weight (0.27 ± 0.15 vs. 0.57 ± 0.32 g, p = 0.02). Microarray analysis revealed that CIT may regulate cell cycle signalling pathway through various cell cycle regulators. ConclusionsIn summary, we provided clinical and experimental evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.

Investigation of Bladder Cancer Cell Response to Cryoablation and Adjunctive Cisplatin Based Cryo/Chemotherapy.

Due to a rising annual incidence of bladder cancer, there is a growing need for development of new strategies for treatment. In 2018, the World Cancer Research Fund and other groups reported that there were ~550,000 new cases worldwide of bladder cancer. It has been further estimated that >200,000 individuals die annually from bladder cancer worldwide. Various treatment options exist. However, many if not all remain suboptimal. While the preferred chemotherapeutic options have changed in the past few years there have been few advances in the bladder cancer medical device field. Cryoablation is now being evaluated as a new option for the treatment of bladder cancer. While several studies have shown cryoablation to be promising for the treatment of bladder cancer, a lack of basic information pertaining to dosing (minimal lethal temperature) necessary to destroy bladder cancer has limited its use as a primary therapeutic option. Concerns with bladder wall perforation and other side effects have also slowed adoption.In an effort to detail the effects of freezing on bladder cancer, two human bladder cancer cell lines, SCaBER and UMUC3, were evaluated in vitro. SCaBER, a basal subtype of muscle invasive bladder cancer, and UMUC3, an intermediate transitional cell carcinoma, are both difficult to treat but are reportedly responsive to most conventional treatments. SCaBER and UMUC3 cells were exposed to a range of freezing temperatures from −10 to −25°C and compared to non-frozen controls. The data show that a single 5 minute freeze to −10°C did not affect cell viability, whereas −15°C and −20°C results in a significant reduction in viability 1 day post freeze to <20%. These populations, however, were able to recover in culture. A complete loss of cell viability was found following a single freeze at −25°C. Application of a repeat (double) freeze resulted in complete cell death at −20°C. In addition to freezing alone, studies investigating the impact of adjunctive low dose (1 μM) cisplatin pre-treatment (30 minutes and 24 hours) in combination with freezing were conducted. The combination of 30 minute cisplatin pre-treatment and mild (−15°C) freezing resulted in complete cell death. This suggests that subclinical doses of cisplatin may be synergistically effective when combined with freezing.In summary, these in vitro results suggest that freezing to temperatures in the range of −20 to 25°C results in a high degree of bladder cancer cell destruction. Further, the data describe a potential combinatorial chemo/cryo therapeutic strategy for the treatment of bladder cancer.

Isoliquiritigenin induces apoptosis through caspases and reactive oxygen species signaling pathways in human bladder cancer cells

Background: Isoliquiritigenin (ISL) is a flavonoid isolated from the roots of various species of licorice plants. Objectives: Mechanisms underlying ISL-induced cell death were investigated in 5637 human bladder cancer cell line. Materials and Methods: Cell viabilities were measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and cell counting kit-8 assay. Cell cycle analysis, caspase activity assay, western blotting, and reactive oxygen species (ROS) assay were also used to investigate the anticancer effects of ISL on 5637 cells. Results: ISL (100–500 μg/ml) inhibited cancer cell proliferation and increased sub-G1 cell cycle phase ratios. ISL-induced cell death resulted in reduced Bcl-2 and increased Bax. ISL also activated caspase-3 and -9 and increased the levels of intracellular ROS generated. In addition, TG100-115 (transient receptor potential [TRP] melastatin 7 inhibitor) and tranilast (TRP vanilloid 2 inhibitor) each exerted a synergistic effect with ISL on ISL-induced apoptosis. Conclusion: These findings suggest that ISL causes apoptosis in 5637 cancer cell line. Therefore, ISL may be a potential anticancer drug for treating bladder cancer and a good anticancer supplement.

A Non-invasive Imageable GFP-expressing Mouse Model of Orthotopic Human Bladder Cancer.

A more realistic mouse model of bladder cancer is necessary to develop effective drugs for the disease. Tumor models enhanced by bright fluorescent-reporter genes to follow the disease in real-time would enhance the ability to accurately predict the efficacy of various therapeutics on this particularly-malignant human cancer. A highly-fluorescent green fluorescent protein (GFP)-expressing bladder cancer model was orthotopically established in nude mice using the UM-UC-3 human bladder-cancer cell line (UM-UC-3-GFP). Fragments from a subcutaneous tumor of UM-UC-3-GFP were surgically implanted into the nude mouse bladder. Non-invasive and intra-vital fluorescence imaging was obtained with a simple imaging box. The GFP-expressing orthotopic bladder tumor was imaged in real-time non-invasively as well as intra-vitally, with the two methods correlating at r=0.99. This is the first non-invasive-fluorescence-imaging orthotopic model of bladder cancer and can be used for rapidly screening novel effective agents for this recalcitrant disease.

MicroRNA-152 suppresses cell proliferation and tumor growth of bladder cancer by targeting KLF5 and MKK7

B ackground: In bladder cancer, miR-152 has been reported to be downregulated and accompanied by high DNA methylation. However, the molecular mechanisms underlying miR-152 in bladder cancer tumorigenesis remain unknown. M ethods: The expressions of miR-152 in human bladder cancer cell lines (J82, 5637, and T24) and a human bladder epithelial cell line (SV-HUC-1) were compared. The expression of miR-152 was observed after adding DNA methylation inhibitor 5-Aza-dC or after knocking down the major DNA methyltransferase DNMT1. The bladder cancer cell viability was determined using an MTT assay, and the colony formation rate was determined using a colony formation assay. The effects of miR-152 overexpression on cell proliferation and tumor growth were observed in vitro and in vivo. The interactions among miR-152, KLF5, and MKK7 were determined using a luciferase reporter assay. R esults: MiR-152 was downregulated in bladder cancer cell lines in comparison with normal ones. The expression of miR-152 was significantly promoted after adding 5-Aza-dC or after knocking down DNMT1. The overexpression of miR-152 suppressed cell proliferation and tumor growth both in vitro and in vivo. MiR-152 inhibited KLF5 and MKK7 expression by binding with 3’ untranslated regions of their mRNAs. Meanwhile, the overexpression of KLF5 induced MKK7 expression by increasing the transcriptional activity of MKK7. The simultaneous overexpression of miR-152 and KLF5 reversed the inhibition of cell proliferation caused by miR-152 overexpression alone. C onclusions: Our findings suggest that miR-152 acts as a tumor suppressor in bladder cancer, and the overexpression of miR-152 may be a potential strategy for treating bladder cancer. K eywords: cell proliferation, bladder cancer, DNA methylation, miR-152, transcription factor

Antitumor effects of MutT homolog 1 inhibitors in human bladder cancer cells.

As standard second-line regimen has not been established for patients who are refractory to or relapse with cisplatin-based chemotherapy, an effective class of novel chemotherapeutic agents is needed for cisplatin-resistant bladder cancer. Recent publications reported that MutT homolog 1 (MTH1) inhibitors suppress tumor growth and induce impressive therapeutic responses in a variety of human cancer cells. Few studies investigated the cytotoxic effects of MTH1 inhibitors in human bladder cancer. Accordingly, we investigated the antitumor effects and the possible molecular mechanisms of MTH1 inhibitors in cisplatin-sensitive (T24) and - resistant (T24R2) human bladder cancer cell lines. These results suggest that TH588 or TH287 may induce cancer cell suppression by off-target effects such as alterations in the expression of apoptosis- and cell cycle-related proteins rather than MTH1 inhibition in cisplatin-sensitive and - resistant bladder cancer cells.Abbreviations: MTH: MutT homolog; ROS: reactive oxygen species; CCK-8: cell counting kit-8; DCFH-DA: dichlorofluorescein diacetate; PARP: poly (ADP-ribose) polymerase.

The modulation study of multiple drug resistance in bladder cancer by curcumin and resveratrol.

Gemcitabine (GCB), which functions via the inhibition of DNA synthesis, is commonly used in the treatment of bladder cancer; however, its response rate is not satisfactory due to the development of drug resistance. The potential for phytochemicals to reverse drug resistance in bladder cancer tumor cells was evaluated. A human bladder cancer cell line, T24, was cultured, and GCB-resistant cells (T24-GCB) were also established. The acquired resistance of T24-GCB to GCB was measured using an MTT assay. The gene expression of ATP-binding cassette (ABC) transporter protein family members was analyzed using reverse transcription-quantitative PCR analysis, and western blotting was performed to verify ABC family protein, cytoplasmic thymidine kinase (TK) and poly (ADP-ribose) polymerase (PARP) expression on whole cell lysates. Subsequently, resveratrol and curcumin were used to evaluate their modulation potential in decreasing the drug resistance of T24-GCB cells to GCB using MTT and migration assays. T24-GCB cells have increased drug resistance ability, with an 18.75-fold higher ID50 value compared with native T24 cells (105 vs. 5.6 nM). T24-GCB cells also exhibit increased cross resistance to mitomycin C and paclitaxel. The mRNA expression of ABCC2 in T24-GCB cells increased compared with that in native T24 cells. Via western blot analysis, it was determined that the expression of ABCC2 protein was also increased in T24-GCB cells. Conversely, the expression of ABCB1, ABCG2, deoxycytidine kinase (DCK), TK1 and TK2 decreased. Following curcumin and resveratrol treatment alone or combined with GCB, additive cytotoxic enhancement was observed, and the migratory abilities of T24-GCB cells were significantly decreased. Western blot analysis revealed that ABCC2 protein expression increased, and DCK, TK1 and TK2 expression decreased following co-treatment of T24-GCB cells with GCB + curcumin or resveratrol compared with GCB alone. Of note, there was a marked increase in cleaved-PARP expression in T24-GCB cells treated with a combination of GCB + curcumin or resveratrol. Both curcumin and resveratrol could reverse the drug resistance of T24-GCB cells in an additive pattern though PARP enhancement without changes in ABCC2 and DCK, TK1 and TK2 expression.

Circular RNA circ-Foxo3 induced cell apoptosis in urothelial carcinoma via interaction with miR-191-5p.

BackgroundCircular RNAs (circRNAs) play a critical role in cancer. Emerging evidence has shown circ-Foxo3, a circRNA, was dysregulated in a variety of tumor types. However, the exact role of circ-Foxo3 in bladder cancer has never been studied.MethodsWe measured the expression level of circ-Foxo3 in human and murine bladder cancer tissues and in various human bladder cancer cell lines. We induced bladder cancer in mice by a carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). circ-Foxo3 was overexpressed in mice by lentiviral gene transfer and in cultured cells via overexpression plasmid. The effect of circ-Foxo3 on apoptosis was examined via apoptotic marker staining, Western blot, and flow cytometry. We further characterized the interaction between circ-Foxo3 and miR-191 and its functional impact on bladder cancer cells.Resultscirc-Foxo3 was downregulated in bladder cancer in vivo and in vitro, and was upregulated in response to apoptotic stress. Overexpression of circ-Foxo3 promoted bladder cancer cell apoptosis in BBN mice and in human bladder cancer cell lines. miR-191-5p suppressed circ-Foxo3 expression and the pro-apoptotic effect of circ-Foxo3 in bladder cancer cells via directly targeting the 3ʹ-untranslated region (3ʹ-UTR) of circ-Foxo3.Conclusioncirc-Foxo3 was downregulated in bladder cancer in vivo and in vitro, and promoted bladder cancer apoptosis via direct interaction with miR-191. circ-Foxo3 could be a potential therapeutic target for bladder cancer.

61P - Autophagy inhibition enhances leflunomide-induced cytotoxicity in human bladder cancer cells

BackgroundDihydroorotate dehydrogenase (DHODH) is one of the essential enzymes in the de novo biosynthesis of pyrimidine and might be a potential therapeutic target for cancer suppress. The anti-proliferative and apoptosis-inducing effects of leflunomide, a potent DHODH blocker, have been demonstrated in multiple human cancers. This study aims to investigate the cytostatic effects of leflunomide on bladder cancer and the involved mechanism. MethodsTwo human bladder cancer cell lines, T24 and 5637, were used in this study. After incubation with varied doses of leflunomide, the cell viability, apoptosis and cell cycle assay were determined with MTS, cell colony assay and flow cytometry. Western blot was used to evaluate the expression changes of cleaved-PARP, proteins involved in Akt/mTOR/P70S6K signaling pathway and cell autophagy pathway. AVO stain assay was performed to detect the autophagosome. Moreover, the cytostatic effects of leflunomide were further investigated after the modulation of cell autophagy with autophagy agonist rapamycin and inhibitor chloroquine. ResultsOur data demonstrated that leflunomide markedly inhibited the growth of both bladder cancer cells via inducing cell apoptosis and cell cycle arrest in S phase in a time- and dose-dependent manner. After leflunomide treatment, the phosphorylation levels of Akt, mTOR and p70S6K proteins in both cells were significantly down-regulated. Furthermore, AVO stain assay revealed the decline of autophagosome under the incubation of leflunomide. Modulation of autophagy with rapamycin and chloroquine observably attenuated and enhanced the cytostatic effects of leflunomide, respectively. ConclusionsLeflunomide significantly reduced the cell viability of bladder cancer cells via Akt/mTOR/P70S6K signaling pathway. In addition, cell autophagy was demonstrated to be involved, combination leflunomide with autophagy modifier exerted enhanced antitumor effects in bladder cancer, which offered novel ideas for bladder cancer treatment. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Circular RNA SMARCA5 is overexpressed and promotes cell proliferation, migration as well as invasion while inhibits cell apoptosis in bladder cancer

Background: This study aimed to investigate the function of circular RNA SMARCA5 (circ-SMARCA5) on cell proliferation, apoptosis, migration and invasion in bladder cancer. Methods: Ten pairs of human bladder cancer tissue and adjacent tissue, five human bladder cancer cell lines (including TCCSUP, 5637, J82, UM-UC-3 and T-24) and normal human urothelial cell line SV-HUC-1, were obtained for the detection of circ-SMARCA5. Control overexpression and ShRNA, circ-SMARCA5 overexpression and ShRNA were constructed and transfected into UM-UC-3 cells as Control(+), Control(−), Circ-SMARCA5(+) and Circ-SMARCA5(−) groups. The role of circ-SMARCA5 was investigated in terms of cellular proliferation, apoptosis, migration, and invasion. Results: Circ-SMARCA5 was overexpressed in tumor tissue compared to paired adjacent tissue and it was also overexpressed in TCCSUP, 5637, J82 and UM-UC-3 cells compared to normal human urothelial cell line SV-HUC-1. In UM-UC-3 cells, cell proliferation ability, migration rate and invasion cell count were increased in Circ-SMARCA5(+) group compared to Control(+) group, while reduced in Circ-SMARCA5(−) group compared to Control(−) group. Regarding the cell apoptosis, apoptosis rate and apoptotic protein C-Caspase 3 expression were decreased in Circ-SMARCA5(+) group than those in Control(+) group but raised in Circ-SMARCA5(−) group compared to Control(−) group, meanwhile, the anti-apoptotic protein Bcl-2 expression was elevated in Circ-SMARCA5(+) group than that in Control(+) group but reduced in Circ-SMARCA5(−) group compared to Control(−) group. Conclusions: Circ-SMARCA5 is overexpressed, and promotes cell proliferation, migration and invasion, but represses apoptosis in bladder cancer.

Tumor-suppressor microRNA-139-5p restrains bladder cancer cell line ECV-304 properties via targeting Connexin 43.

Background:In our previous paper, we demonstrated that Connexin 43 (CX43) was highly expressed in bladder cancer (BC) tissues. But the molecular mechanism about microRNAs (miRNAs) regulation upstream of CX43 in BC has not been well elucidated and remains to be further studied. MicroRNA-139-5p (miR-139-5p) is a tumor suppressor in progression of multifarious cancers including BC. Nevertheless, the underlying mechanisms of CX43/miR-139-5p in tumorigenesis of BC are still not well illustrated. The specific objective of our study was to inquiry the effect of CX43/miR-139-5p on BC progression and its underlying mechanism.Methods:The bioinformatics analysis softwares were applied to predict the miRNAs in the upstream of CX43. First, the expression levels of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal) were investigated using the data from The Cancer Genome Atlas database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of miR-139-5p in three human BC cell lines 5637, T24, ECV-304 and a human bladder epithelial immortalized cell line SV-HUC-1 (normal control). Then si-CX43, si-control, miR-139-5p mimic, and its negative control (NC) were transfected into BC cell line ECV-304. The relationship of miR-139-5p and CX43 was analyzed by dual-luciferase reporter assay. The qRT-PCR and Western blotting were used to test the mRNA and protein expression level of CX43. The proliferation of ECV-304 and T24 cells were examined by cell counting kit-8. The migration and invasion of ECV-304 cells were tested by transwell assay. To determine whether miR-139-5p would affect cell proliferation, migration and invasion by targeting CX43, we executed the rescue assay. The comparison between two groups was analyzed by Student's t test, and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.Results:The expression of miR-139-5p was remarkably down-regulated in BC tissues (tumor vs. normal, 2.286 ± 0.017 vs. 3.211 ± 0.034, t = 11.540, P < 0.0001) and cell lines (P < 0.01 in all BC cell lines). Besides, we also indicated that over-expression of miR-139-5p reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005). Moreover, miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. Furthermore, the relative luciferase activity of CX43-wild type construct was distinctly lessened by up-regulation of miR-139-5p (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.916 ± 0.063 vs. 0.356 ± 0.048, t = 7.085, P = 0.002), nevertheless the activity of CX43-mutant type construct was untouched (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.918 ± 0.057 vs. 0.878 ± 0.039, t = 0.577, P = 0.595). Finally, the rescue assay revealed that CX43 deletion enhanced the depressor effect of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014).Conclusion:MiR-139-5p, as a tumor-suppressor, repressed cell proliferation, invasion, and migration in BC, which might be achieved by regulating CX43.

Oncolytic Immunotherapy for Bladder Cancer Using Coxsackie A21 Virus: Using a Bladder Tumor Precision-Cut Slice Model System to Assess Viral Efficacy.

Oncolytic viruses are anticancer agents that selectively target and kill cancer cells by direct lysis, while at the same time stimulating a tumor antigen-specific adaptive immune response. These promising therapeutic agents target multiple cancers and have already proven to be an effective treatment option for solid malignancies. One such agent, T-Vec (Talimogene laherparepvec) has been licensed and is in routine clinical use for treatment of malignant melanoma.Non-muscle invasive bladder cancer (NMIBC) is an ideal potential target for oncolytic immunotherapy as locally instilled live biological therapy using Bacille Calmette-Guerin (BCG) is already well established in the clinical setting. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. We have investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. In addition to cell lines and an animal model a key component of our studies into oncolytic immunotherapy for bladder cancer was the use of a bladder tumor precision slice preclinical model system which represents tumor architecture, heterogeneity, and the complexity of a tumor in vitro. Results seen in cell lines were reflected in the tumor slice model whereby levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. In this chapter we demonstrate the utility of the precision cut tumor slice model as a unique organotypic model to test oncolytic viruses. We will describe how to prepare and slice the tumor using a vibrating microtome together with the optimum culture and conditions for treatment.

Circular RNA SMARCA5 is overexpressed and promotes cell proliferation, migration as well as invasion while inhibits cell apoptosis in bladder cancer.

BackgroundThis study aimed to investigate the function of circular RNA SMARCA5 (circ-SMARCA5) on cell proliferation, apoptosis, migration and invasion in bladder cancer.MethodsTen pairs of human bladder cancer tissue and adjacent tissue, five human bladder cancer cell lines (including TCCSUP, 5637, J82, UM-UC-3 and T-24) and normal human urothelial cell line SV-HUC-1, were obtained for the detection of circ-SMARCA5. Control overexpression and ShRNA, circ-SMARCA5 overexpression and ShRNA were constructed and transfected into UM-UC-3 cells as Control(+), Control(−), Circ-SMARCA5(+) and Circ-SMARCA5(−) groups. The role of circ-SMARCA5 was investigated in terms of cellular proliferation, apoptosis, migration, and invasion.ResultsCirc-SMARCA5 was overexpressed in tumor tissue compared to paired adjacent tissue and it was also overexpressed in TCCSUP, 5637, J82 and UM-UC-3 cells compared to normal human urothelial cell line SV-HUC-1. In UM-UC-3 cells, cell proliferation ability, migration rate and invasion cell count were increased in Circ-SMARCA5(+) group compared to Control(+) group, while reduced in Circ-SMARCA5(−) group compared to Control(−) group. Regarding the cell apoptosis, apoptosis rate and apoptotic protein C-Caspase 3 expression were decreased in Circ-SMARCA5(+) group than those in Control(+) group but raised in Circ-SMARCA5(−) group compared to Control(−) group, meanwhile, the anti-apoptotic protein Bcl-2 expression was elevated in Circ-SMARCA5(+) group than that in Control(+) group but reduced in Circ-SMARCA5(−) group compared to Control(−) group.ConclusionsCirc-SMARCA5 is overexpressed, and promotes cell proliferation, migration and invasion, but represses apoptosis in bladder cancer.

Detection of tyrosine kinase inhibitors-induced COX-2 expression in bladder cancer by fluorocoxib A.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo. The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro. The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro. In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo. In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.